The topic of bone formation regulation and bone-related disorders has recently gained considerable attention; for example in the women's health area, there has been a particular focus on the bone-related disorder of osteoporosis. Throughout life, there is a constant remodeling of skeletal bone. In this remodeling process, there is a fragile balance between bone formation by osteoblasts and subsequent bone resorption by osteoclasts.
As a normal part of the aging process the bone matrix undergoes various structural changes, the nature of which remains not fully determined. The majority of studies on age-related changes in human bone have been directed towards elucidating changes in bone on a morphological level or by quantitatively comparing rates of bone loss. Identification of the mechanisms involved in bone disorders is crucial for the understanding of bone physiology and bone disorders. While numerous genes and gene families and the polypeptides encoded by them that participate in the regulation of bone cells have been identified and cloned, their functions have not been clearly delineated due to the complexities of the bone formation pathways.
The WNT Gene Family
One group of genes and the proteins encoded by them that play an important role in regulating cellular development is the Wnt family of glycoproteins. Wnt proteins are a family of growth factors consisting of more than a dozen structurally related molecules and are involved in the regulation of fundamental biological processes like apoptosis, embryogenesis, organogenesis, morphogenesis and tumorigenesis (reviewed in Nusse and Varmus 1992 Cell 69: 1073–1087). These polypeptides are multipotent factors and have similar biological activities to other secretory proteins like transforming growth factor (TGF)-β, fibroblast growth factors (FGFs), nerve growth factor (NGF) and bone morphogenetic proteins (BMPs). One member of the Wnt growth factor family termed Wnt-x, is preferentially expressed in bone tissue and in bone-derived cells and appears to be involved in maintaining the mature osteoblast (bone-forming cell) phenotype (PCT/US94/14708; WO 95/17416).
The Frizzled Family of Proteins
Studies indicate that certain Wnt proteins interact with a family of proteins named “Frizzled” that act as receptors for Wnt proteins or as components of a Wnt receptor complex (reviewed in Moon et al. 1997 Cell 88: 725–728 and Barth et al. 1997 Curr. Opin. Cell Biol. 9: 683–690). Frizzled proteins contain an amino terminal signal sequence for secretion, a cysteine-rich domain (CRD) that is thought to bind Wnt, seven putative transmembrane domains that resemble a G-protein coupled receptor, and a cytoplasmic carboxyl terminus.
The discovery of the first secreted frizzled-related protein (SFRP) was reported by Hoang et al. in 1996 (J. Biol. Chem. 271: 26131–26137). This protein, which was called “Frzb” for frizzled motif in bone development, was purified and cloned from bovine articular cartilage extracts based on its ability to stimulate in vivo chondrogenic activity in rats. The human homologue of the bovine gene was also cloned. However, unlike the frizzled proteins, Frzb did not contain a serpentine transmembrane domain. Thus, this new member of the frizzled family appeared to be a secreted receptor for Wnt. The Frzb cDNA encoded for a 325 aa/36,000 Dalton protein and was predominantly expressed in the appendicular skeleton. The highest level of expression was in developing long bones and corresponded to epiphyseal chondroblasts; expression then declined and disappeared toward the ossification center.
Recent studies indicate SFRPs participate in apoptosis and thus some SFRPs have been identified as “SARPs” for secreted apoptosis related proteins. Additional members of the SFRP family have also recently been identified and shown to be antagonists of Wnt action. There are currently at least five known human SFRP/SARP genes: SFRP-1/FrzA/FRP-1/SARP-2, SFRP-2/SDF-5/SARP-1, SFRP-3/Frzb-1/FrzB/Fritz, SFRP-4 and SFRP-5/SARP-3 (Leimeister et al 1998 Mechanisms of Development 75: 29–42, which sequences of this reference are incorporated herein). Although the precise role that SARPs/SFRPs play in apoptosis is not yet clear, these proteins appear to either suppress or enhance the programmed cell death process.
In summary, a great need exists for the definitive identification of targets for the treatment of bone disorders, including bone resorption disorders such as osteoporosis and for regulation of bone formation in humans.